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Method for preparing warm start Taq enzyme

A hot-start, animal technology, applied in biochemical equipment and methods, chemical instruments and methods, enzymes, etc., can solve the problems of non-specific amplification, prone to clonal mismatches, etc., to improve specificity and reduce clonal mismatches , The effect of strong anti-freeze and thaw ability

Inactive Publication Date: 2017-09-01
GUANGZHOU SUPBIO BIO TECH & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a real-time hot-start Taq enzyme and its preparation method to solve the problems of clone mismatch and non-specific amplification that are prone to occur in the PCR reaction

Method used

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  • Method for preparing warm start Taq enzyme
  • Method for preparing warm start Taq enzyme
  • Method for preparing warm start Taq enzyme

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Embodiment 1

[0031] Embodiment 1 A kind of preparation method of hot start Taq enzyme

[0032] A preparation method of hot start Taq enzyme, its steps are as follows:

[0033] (1) Obtain Taq enzyme: the Taq enzyme used in this embodiment is the Taq enzyme whose Genbank number is gi|155128, and its obtaining method is:

[0034] 1) Design primers: design the following PCR amplification primers according to its DNA sequence

[0035] Upstream primer, its sequence is shown in SEQ ID NO: 1: 5'-GGC GAT GCT GCC CCT C-3',

[0036] The downstream primer has a sequence as shown in SEQ ID NO: 2: 5'-GTC GAC ATC ACT CCT TGG CG-3';

[0037]2) PCR amplification: using Thermus aquaticus as a template, using the upstream primer and downstream primer described in step 1), the coding gene of Taq enzyme was obtained by conventional PCR amplification;

[0038] The PCR amplification program used was: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds,...

Embodiment 2

[0043] Embodiment 2 A kind of preparation method of hot start Taq enzyme

[0044] A preparation method of hot start Taq enzyme, its steps are as follows:

[0045] (1) obtain Taq enzyme: with embodiment 1;

[0046] (2) Preparation of Taq enzyme nanobody: same as Example 1;

[0047] (3) Preparation of hot-start Taq enzyme: the Taq enzyme obtained in step (1) and the Taq enzyme nanobody obtained in step (3) were mixed at a molar ratio of 0.9:1, and incubated at 40°C for 2h to make the antigen-antibody The complex is formed, and the hot start Taq enzyme is obtained.

Embodiment 3

[0048] Embodiment 3 A kind of preparation method of hot start Taq enzyme

[0049] A preparation method of hot start Taq enzyme, its steps are as follows:

[0050] (1) Obtain Taq enzyme: purchased from Shenzhen Faipeng Biological Co., Ltd. (article number MD001);

[0051] (2) Preparation of Taq enzyme nanobody: same as Example 1;

[0052] (3) Preparation of hot-start Taq enzyme: the Taq enzyme obtained in step (1) and the Taq enzyme nanobody obtained in step (3) were mixed in a molar ratio of 1:1.1, and incubated at 35°C for 1.5h to make the antigen The antibody forms a complex, ie the hot start Taq enzyme.

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Abstract

The invention discloses a real-time warm start Taq enzyme and a preparation method thereof. The real-time warm start Taq enzyme is an antigen-antibody complex of a Taq enzyme and a Taq enzyme nano antibody through antigen-antibody reaction, wherein the Taq enzyme nano antibody is derived from but not limited by animals which can generate nano antibodies through Taq enzyme immunoassay. By forming the antigen-antibody complex, the activity of the Taq enzyme can be released only at relatively high temperature, warm start can be achieved, the specificity, the reliability and the sensitivity of the Taq enzyme are improved, generation of non-specific amplification is avoided, and the Taq enzyme is capable of reducing clone mispairing to a very large extent and achieving efficient specific amplification when being compared with similar products. The nano antibody has high affinity and high stability, is capable of greatly improving specificity, sensitivity and amplification efficiency of warm start PCR (Polymerase Chain Reaction), and is applicable to various PCR reactions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a Taq enzyme, in particular to a method for preparing a hot-start Taq enzyme. Background technique [0002] Polymerase chain reaction (PCR), a technique for amplifying specific DNA fragments in vitro, is an important tool in the fields of molecular biology and medicine. The DNA polymerase used in PCR is mainly Taq enzyme. With the promotion and application of PCR technology, the research on the properties of Taq enzyme is becoming more and more important. [0003] Taq enzyme is a thermostable DNA polymerase isolated from a Thermus aquaticus. The enzyme is thermally stable, but still has a certain polymerase activity at 20°C to 37°C, and is prone to non-specific amplification and primer-dimers. In order to solve these problems, people invented hot-start PCR, which increases the optimal reaction temperature of Taq enzyme through various ways to achieve the purp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C07K16/40
CPCC07K16/40C07K2317/22C07K2317/569C12N9/1252C12Y207/07007
Inventor 林志豪张晓玮彭春梅张嘉邓可基李海茵谢丽娟李家导乐小炎罗园香张新陈观芝陈凤英林敏深石壮壮林若琳王星王法余培煜莫静嫣
Owner GUANGZHOU SUPBIO BIO TECH & SCI
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